Isolation and Characterization of Poeciguamerin, a Peptide with Dual Analgesic and Anti-Thrombotic Activity from the Poecilobdella manillensis Leech

Abstract

When Poecilobdella manillensis attacks its prey, the prey bleeds profusely but feels little pain. We and other research teams have identified several anticoagulant molecules in the saliva of P. manillensis, but the substance that produces the paralyzing effect in P. manillensis is not known. In this study, we successfully isolated, purified, and identified a serine protease inhibitor containing an antistasin-like domain from the salivary secretions of P. manillensis. This peptide (named poeciguamerin) significantly inhibited elastase activity and slightly inhibited FXIIa and kallikrein activity, but had no effect on FXa, trypsin, or thrombin activity. Furthermore, poeciguamerin exhibited analgesic activity in the foot-licking and tail-withdrawal mouse models and anticoagulant activity in the FeCl₃-induced carotid artery thrombosis mouse model. In this study, poeciguamerin was found to be a promising elastase inhibitor with potent analgesic and antithrombotic activity for the inhibition of pain and thrombosis after surgery or in inflammatory conditions.

Keywords: Poecilobdella manillensis; analgesic; anti-thrombotic; poeciguamerin; thrombus formation.

Figure 1

Isolation and purification of poeciguamerin from P. manillensis salivary gland secretions. (A) A Sephadex G-50 dextran gel filtration column connected to a fully automated partial collector was used for sample collection, with a flow rate of 0.3 mL/min and one tube collected every 10 min. Sample absorbance at 280 nm and 215 nm was measured in alternate tubes using a spectrophotometer. The components of each peak were collected and integrated based on absorbance values. (B) Elastase activity inhibition by the three peak fractions was detected. (C) The protein peak with elastase activity inhibition was separated and purified using liquid chromatography. An RP-HPLC C8 column with a UV detector was used to detect the light absorbance of the sample at 280 nm and 215 nm. The dashed line indicates a linear gradient of acetonitrile from 10% to 40% over 30 min. (D) Elastase activity inhibition was detected for the four peak components. (E) Collected peaks were further purified by monitoring at 280 nm using a Mono S^TM 5/50 GL column connected to an AKTA FPLC system. (F) Elastase activity inhibition was detected for the four peak fractions.

Figure 2

Primary structure of poeciguamerin. (A) Determination of amino acid sequence of poeciguamerin by the Edman degradation method and local blast with our transcriptome data (data not shown in the text) of the salivary glands of P. manillensis. C (Cysteine) is marked in red, and the green part represents the predicted Antistasin-like domain. (B) Amino acid sequences of different proteins, including poeciguamerin (P. manillensis), guamerin [18] (Hirudo nipponia, P46443), gelin [34] (P. manillensis, AAB27871.1), poecistasin [16] (P. manillensis), hirustasin [35] (P. manillensis, P80302), and piguamerin [17] (P. manillensis, P81499), were compared for multiple sequence alignment. Shaded areas indicate conserved sequences. Shaded areas indicate conserved sequences. The degree of amino acid sequence conservation is marked by color, and the colors from low to high are blue, orange, and red, respectively. * represents the absence of amino acids in that position. (C) Protein size analysis of poeciguamerin by MALDI-TOF-MS.

Figure 3

Effects of poeciguamerin on elastase and coagulation. Effects of natural poeciguamerin on elastase (A), FXIIa (B), kallikrein (C), FXa (D), trypsin (E), and thrombin (F) enzymatic activities.

Figure 4

Ki values for the inhibition of elastase (A), FXIIa (B), and kallikrein (C) enzymatic activities by poeciguamerin, calculated using Dixon plot curves [36].

Figure 5

Effects of poeciguamerin on elastase or hot water bath-induced pain. Control mice were injected with 25 μL of elastase (6 mg/kg) in the sole of the right hind paw, while experimental mice were intravenously injected with poeciguamerin (12 mg/kg) 10 min prior to the elastase injection. The total time mice spent licking the paw was recorded during the I phase (0–10 min) (A) and the II phase (10–30 min) (B). n = 5 per group. Data are expressed as mean ± SEM and were subjected to an unpaired t-test, *** p < 0.001. In total, 20 mice with tail retraction latencies of 4 to 8 s were selected, 5 mice per group. Mice were injected with poeciguamerin (0, 6, and 12 mg/kg) or morphine (0.2 mg/kg) into the tail vein prior to stimulation of the tail with a hot water bath for 10 min. The tails of the mice were stretched in hot water at 45 °C and the time of the tail flick was recorded (C). n = 5 per group. Data are expressed as mean ± SEM and were subjected to an unpaired t-test, *** p < 0.001.

Figure 6

Effects of poeciguamerin on thrombosis in a mouse model of FeCl₃-induced carotid artery injury. Different concentrations of recombinant poeciguamerin (0, 1.25, 2.5 mg/kg) and sodium heparin (20 mg/kg) were injected into the tail vein 10 min prior to the surgical procedure to establish the model. After exposure of the carotid artery, blood flow was monitored for 0, 4, 8, 12, 16, 20, 24, and 28 min after applying 10% FeCl₃ to the exposed artery. (A) Image of carotid flow from laser scatter perfusion imaging. (B) Quantitative carotid flow data of the perfusion unit at the ROI. n = 3 per group. Data are expressed as mean ± SEM.